(1) Technical Field
The present invention relates to a light detecting apparatus for detecting light intensity of fluorescence, reflected light, transmitted light, etc., obtained by illuminating an object under observation with light, and mainly relates to an apparatus which detects fluorescence distribution when excitation light is irradiated, in particular, relating to a fluorescence detecting apparatus for detecting 2-dimensional separation and development of a fluorescence-labeled sample by electrophoresis etc.
(2) Related Art and Other Considerations
Generally, methods for obtaining information of an object to be observed by an optical detecting means that illuminates an object under observation with light have been used since a long time ago, for various purposes in various technical fields. Also there have been developed and used various kinds of light detecting devices which collect fluorescence, reflected light etc., emitted from an object under observation by illumination of light, onto a photo-electric transducer and detect light intensity as electric signals to thereby obtain information on the object under observation.
In detection of light intensity, detection accuracy can be improved by use of optical means such as light-separating devices, optical filters etc., that remove components of wavelengths and/or polarized light unnecessary for detection, from the light emitted from an object of study when the object is illuminated.
In particular, when the object under observation is a fluorescent material, it is possible to detect weak fluorescence because, in general, the fluorescence to be detected is different in wavelength from the excitation light for illumination. Though not particularly limited to the field of fluorescence detection, as one example of conventional light detection apparatuses, there are fluorescence detecting apparatuses that detect fluorescence distribution when excitation light is irradiated. Of these, some of fluorescence detecting apparatuses that detect 2-dimensional separation and development of a fluorescence-labeled sample by electrophoresis etc., are demanded to have detection performance with high sensitivity over a wide dynamic range in order to deal with detection from an extremely small amount of fluorescent dyes to a certain amount of fluorescent dyes.
As the conventional fluorescence detecting apparatuses, two kinds of apparatuses have been mainly used, namely, scan-type fluorescence detecting apparatus and camera-type fluorescence detecting apparatus. With regard to the scan-type fluorescence detecting apparatus, a laser beam of excitation light is irradiated to the detection point so that the fluorescence from the irradiated point is detected by a PMT (photomultiplier tube), and this irradiation and detection is scanned throughout the object to be observed to thereby obtain fluorescence information of a 2-dimensional area (Japanese Patent Application Laid-open H09-210907).
On the other hand, with regard to the camera-type fluorescence detecting apparatus, excitation light is irradiated on the area to be detected by an area light source so that fluorescence from a wide area is captured all at once by a CCD (charge coupled devices) area image sensor to thereby obtain fluorescence information of a 2-dimensional region. In most cases, CCD is cooled to reduce dark current noise and achieve high sensitivity.
For example, the sample fractionated in a 2-dimensional region by 2-dimensional electrophoresis using a gel plate etc., is processed through a dyeing step using fluorescent dyes before or after electrophoresis, and is detected as a distribution of fluorescence spots by the fluorescence detecting apparatus.
FIG. 1 is a view showing a conventional camera-type fluorescence detecting apparatus.
Light from a lamp source 90 passes through an optical filter 91 which transmits selected wavelengths for excitation light, a lens 92 or the like for condensing light on a predetermined position, to be irradiated on a sample surface 93. A fluorescently labeled sample emits fluorescence as it is irradiated with excitation light, and the fluorescence passes through an optical filter 94 which transmits the fluorescence wavelengths only and a lens 95 or the like for focusing light on and around the sample surface, and enters a CCD area image sensor 96. Since the distribution information of the fluorescent spots of the sample is obtained as an image as a whole by CCD area image sensor 96, it is possible to perform detection over the 2-dimensional area in a relatively short period.
In a 2-dimensional separation and observation of proteins or the like, capability of separation and detection of an extremely trace amount of sample is demanded. In order to obtain a more amount of information from 2-dimensional separation, it is considered that not only conventional static observation after separation but also a dynamic area-observation for detecting the continuous separation process by electrophoresis or the like is needed.
However, in the aforementioned conventional fluorescence detecting apparatus configurations, it has been impossible for any of the apparatus configurations to accomplish both the purpose of detecting separation of an extremely trace amount of a sample and the purpose of detecting a continuous separation process of the sample at the same time.
Since in the scan-type fluorescence detecting apparatus spot detection is performed using irradiation of a laser beam of excitation light, this configuration has extremely little adverse effect from light other than the irradiated point, hence is excellent in detection sensitivity. However, it takes long time for detection because detection is performed basically one spot at a time and scanning needs to be performed over the entire 2-dimensional area. Accordingly, it is practically impossible to perform a continuous process of separation except when separation and change of the sample is very slow.
In contrast, in the camera-type fluorescence detecting apparatus, it is possible to detect the fluorescence information of the image-taken area as a whole at almost the same time since the 2-dimensional area is shot at once, and hence it is possible to detect a continuous process of separation by taking images every predetermined period. However, since in this configuration the image pickup area is illuminated as a whole by an area light source, it is impossible to detect and separate weak fluorescence if there are a multiple number of fluorescent spots in the area under observation because light of fluorescence wavelengths spreads over the whole area hence increases background intensity. Further, this tendency becomes more serious in the area proximity to a bright spot that emits strong fluorescence, hence weak fluorescent spots become hard to separate, getting buried.
In conclusion, 2-dimensional separation and observation suffers the problem in that it was practically impossible for the conventional fluorescence detecting apparatus configurations to perform continuous detection of fluorescence in the process in which faint fluorescent spots from an extremely trace amount of sample become fractionated.